Mycoplasma genitalium has recently been implicated in both male and female reproductive tract infections and may account for up to 25% of all cases of non-gonococcal urethritis (NGU). This organism was independently associated with NGU in virtually all studies using DNA amplification methods and, given the strength of this association, it is now appropriate to identify effective treatment regimens for M. genitaliumassociated urethritis. Although small-scale studies have provided some limited information on failure rates for selected antibiotic regimens treating M. genitalium infections, to date, no systematic, randomized treatment trials have been conducted. Further, although antimicrobial susceptibilities of M. genitalium have been determined for a small number of isolates, those strains were collected 10-20 years ago, and thus may not reflect the current antimicrobial profiles of this bacterium. Determining antimicrobial susceptibilities for M. genitalium is complicated by the fastidious nature of this organism, which makes its isolation and growth (necessary components of the antimicrobial susceptibility testing process) challenging. Therefore, in Aim 1 of this project, we propose to improve techniques for isolation and cultivation of M. genitalium, using our quantitative PCR assay to measure growth. With these enhanced methods, we will then determine the antibiotic susceptibility of recently collected clinical strains of M. genitalium. In Aim 2, we will conduct a randomized, double-blinded, placebo-controlled trial to compare the relative effectiveness of azithromycin and doxycycline in eradicating M. genitalium from men with NGU. We will also describe the relationship between antimicrobial resistance and persistent infection by comparing MIC values among patients with persistent detection of M. genitafium at follow-up visits to MIC values in patients who experience microbiologic cure. Finally, in Aim 3, a nested case-control study will compare the proportion of persistent M. genitalium infections among patients experiencing persistent/recurrent symptoms of urethritis (clinical treatment failures or cases) to the proportion of persistent M. genitalium infections among patients experiencing clinical cure (controls). We anticipate that these studies will elucidate the appropriate treatment regimens for this emerging pathogen, as well as further define its role in the etiology of NGU.